Allosteric activation or inhibition of PI3Kγ mediated through conformational changes in the p110γ helical domain

Reviewer #1 (Public Review):

The authors examine signaling factors that differentiate parallel routes to activating phosphoinositide 3-kinase gamma (PI3Kγ). Dissecting the convergent pathways that control PI3Kγ activity is critical because PI3Kγ is a therapeutic target for treating inflammatory disease and cancer. Here, the authors employ a multipronged approach to reveal new aspects for how p84 and p101 pair with p110γ to activate the PI3Kγ heterodimer. The key instigator to this study is a previously reported inhibitory Nanobody, NB7. The hypothesized mechanism for NB7 allosteric inhibition of p84- p110γ was previously proposed to involve blockage of the Ras-binding domain. The authors revise the allosteric inhibition model based on meticulous profiling of various PI3Kγ complex interactions with NB7. In parallel, a cryo-EM-derived model of NB7 bound to the p110γ subunit convincingly reveals a Nanobody interaction pocket involving the helical domain and regulatory motifs of the kinase domain. This revelation shifts the focus to the helical domain, a known target of PKC phosphorylation. While the connections between NB7 interactions and the effects of PKC phosphorylation are sometimes tenuous, it could be argued that the Nanobody served as a tool to reveal the importance of the helical domain to p110γ regulation.

The sites of PKC-mediated p110γ helical domain phosphorylation were unexpectedly inaccessible in the available structural models. Nevertheless, mass spectrometry (MS)-based phosphorylation profiling indicates that PKC can phosphorylate the helical domain of p110γ and p84/p110γ (but not p101/p110γ) in vitro. The authors hypothesize that helical domain dynamics dictate susceptibility to PKC phosphorylation. To explore this notion, carefully executed, rigorous H/D exchange MS (HDX-MS) experiments were performed comparing phosphorylated vs. unphosphorylated p110γ. Notably, this design reveals more about the consequences of p110γ phosphorylation, rather than the mechanisms of p84/p101 promoting/resisting phosphorylation. Nevertheless, HDX-MS is very well suited to exploring secondary structure dynamics, and helical domain phosphorylation strikingly increases dynamics consistent with increased regional accessibility. The increased dynamics also nicely map to the pocket enveloped by the inhibitory NB7 Nanobody.

Ultimately, this study reveals an unexpected p110γ pocket that allows an engineered Nanobody to allosterically inhibit PI3Kγ complexes. The cryo-EM characterization of the interaction inspired an HDX-MS investigation of known sites of phosphorylation in the region. These insights could be linked to differences/convergences of p84 and p101 complex formation and activation of PI3Kγ, and future work may clarify these mechanisms further. The data presented herein will also be useful for broadening the target surface for future therapeutic developments. New allosteric connections between effector binding sites and post-translational modifications are always welcome.

Reviewer #2 (Public Review):

Harris et al. have described the cryo-EM structure of PI3K p110gamma in a complex with a nanobody that inhibits the enzyme. This provided the first structure of full-length of PI3Kgamma in the absence of a regulatory subunit. This nanobody is a potent allosteric inhibitor of the enzyme, and might provide a starting point for developing allosteric, isotype-specific inhibitors of the enzyme. One distinct effect of the nanobody is to greatly decrease the dynamics of the enzyme as shown by HDX-MS, which is consistent with a growing body of observations suggesting that for the whole PI3K superfamily, enzyme activators increase enzyme dynamics.

The most remarkable outcome of the study is that upon observing the site of nanobody binding, the authors searched the literature and found that there was a previous report of a PKCbeta phosphorylation of PI3Kgamma in the helical domain that is near the nanobody binding site. This led the authors to re-examine the consequence of the phosphorylation armed with better structural models and the tools to study the effects of this phosphorylation on enzyme dynamics. They found that the site of phosphorylation is buried in the helical domain, suggesting that a large conformational change would have to take place to enable the phosphorylation. HDX-MS showed that phosphorylation at three sites clustered in the helical domain generate a distinctly different conformation with rapid deuterium exchange. This suggests that the phosphorylation locks the enzyme in a more dynamic state. Their enzyme kinetics show that the phosphorylated, dynamic enzyme is activated.

While this phosphorylation was reported before, the authors have provided a mechanism for why this activates the enzyme, and they have shown why binders that stabilise the helical domain (such as binding to the p101 regulatory subunit and the nanobody) prevent the phosphorylation. It is this insight into the dynamics of the PI3Kgamma that will likely be the long-lasting influence of the work.

The paper is well written and the methods are clear.